Our long-term goal is to understand the molecular mechanisms of BK channel activation. BK-type K+ channels are activated by voltage and intracellular Ca2+. These channels are important in modulating muscle contraction, neural transmission and circadian pacemaker output, and have been shown to associate with hypertension, schizophrenia, epilepsy and paroxysmal dyskinesia. These channels are being pursued as therapeutic targets for neuronal ischemia, trauma and cognitive decline. The voltage sensor and Ca2+ binding sites in BK channels have been identified. However, the structural basis for the coupling between sensors and the activation gate, which are located in different structural domains, still remains elusive. A central question in this crucial step of BK channel gating is how these different structural domains interact with one another to mediate the coupling between the sensors and the activation gate. This proposal is motivated by the long- awaited and recently solved atomic structures of a whole BK channel. These structures offer new insights on fundamental mechanisms of sensor-pore coupling that may differ from the previous understanding. The structure and functional studies lead to a general hypothesis for answering this question: interactions among the voltage sensor domain (VSD), the cytosolic domain (CTD), and the pore-gate domain (PGD) all contribute to the sensor-pore coupling. We propose three specific aims to examine three key aspects of this hypothesis: 1) the VSD-PGD interactions in VSD-pore coupling, 2) the VSD-CTD interactions in both VSD-pore and Ca2+- pore couplings, and 3) the role of a peptide linker between PGD and CTD in both the VSD-pore and Ca2+-pore couplings. We will use novel structure based screening methods to identify compounds that modulate channel function. These compounds and mutations in the channel protein will serve as probes to indicate the structural motifs that are key to the sensor-pore coupling in BK channels. Threading the biophysics of mutations and modulators onto the channel structures will allow us to understand the molecular mechanisms of how physiological stimuli open BK channels. The results of these studies will identify novel binding sites, chemical cores and new mechanisms of altering channel function by drugs, which will directly help drug development targeting BK channels. Our studies on BK channels may provide insights for the understanding of other ion channels that share similar structural and functional characteristics.